Technical Paper

Microbial Detection in Surface Waters

Submitted To

2013 Academic Year NSF AY-REU Program

Part ofNSF Type I STEP Grant

Sponsored ByThe National Science Foundation

Grant ID No:

College of Engineering and Applied SciencesUniversity of Cincinnati

Cincinnati, Ohio

Prepared By:

Jarod Gregory, Pre-Junior, ACCEND Chemical Engineering B.S., Environmental Engineering M.S.Jonathon Cannell, Pre-Junior, ACCEND Chemical Engineering B.S., M.B.A.

Reviewed By:

______________________________________________

Dr. Lilit YeghiazarianREU Project MentorAssistant Professor

School of Biomedical, Chemical, and Environmental EngineeringUniversity of Cincinnati

September 13 – December 5 2013

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Developing a Hydrogel-Based Microbial Biosensor

By Jarod Gregorya, Jonathon Cannellb, Lilit Yeghiazarian*, and Vasile Nistor*

Keywords: Hydrogel, Biosensor, E. coli, Cryptosporidium, Acriflavine

*Corresponding Authors

Drs. Lilit Yeghiazarian and Vasile NistorSchool of Biomedical, Chemical, and Environmental EngineeringUniversity of Cincinnati2600 Clifton Ave., Cincinnati, OH 45221

aJarod GregoryACCEND – Chemical Engineering B.S., Environmental Engineering M.S., Class of 2015School of Biomedical, Chemical, and Environmental EngineeringUniversity of Cincinnati2600 Clifton Ave., Cincinnati, OH 45221

bJonathon CannellACCEND – Chemical Engineering B.S., M.B.A., Class of 2015School of Biomedical, Chemical, and Environmental EngineeringUniversity of Cincinnati2600 Clifton Ave., Cincinnati, OH 45221

Abstract

Microbial contamination of surface waters is an important economical, health-related, and engineering issue facing the U.S. and countries throughout the world. The absence of timely detection of contamination compounds this issue, and the need for real-time contamination that can be used to dynamically map microbe transport is an unnecessary obstacle in the path to protecting our surface waters. This paper outlines the ability to conjugate microbe-capturing primary antibodies to a versatile material such as a hydrogel, as well as the method to detect primary antibody conjugation. The method of conjugating – using an intermediate cationic molecule that is adsorbed by the hydrogel – sets the foundation for a new technological platform for attaching useful molecules or tools to hydrogels.

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Introduction

Microbe Contamination Detection

Water is perhaps the most precious resource on Earth, as people across the world struggle daily with inadequate access to and protection of clean water. It is easy to associate the issue of water quality with third-world countries, but the protection of water resources is an emerging political, economical, environmental, and health-related issue in the United States. Waterborne pathogens are one of the leading causes of water contamination in the U.S. Approximately 93,000 miles of rivers and streams in our country have elevated bacterial levels (EPA 2000). These pathogens cause sickness and sometimes death in vulnerable populations. The CDC estimates that over 900,000 illnesses and as many as 900 deaths are caused by waterborne pathogens each year (CDC 2008).

The problem of water contamination is compounded by our current inability to detect waterborne pathogens in a timely manner. The predominant methods of detection of these pathogens are time-intensive, usually involving a 24-hour incubation period as part of the detection mechanism. This makes it nearly impossible to have a true grasp on when and where surface waters are contaminated with harmful pathogens. Therefore, there is a critical need to develop methods for microbial detection in real- or near-real time, so that adequate measures can be implemented to reduce human exposure to contaminated waters. The experiments outlined in this paper prove that hydrogels are capable of attaching to primary antibodies for Escherichia coli (E. coli) and cryptosporidium. This capability may be used in an effort to create a mobile and versatile biosensor.

Hydrogels

Hydrogels are hydrophilic polymer networks that consist primarily of water. The hydrogels synthesized in these experiments were poly(N-isopropylacrylamide) (PNIPAM) hydrogels that use Laponite-XLG as a polymer cross-linker. These particular hydrogels undergo a dramatic volume phase transition above approximately 33 oC (Arora, et. al. 2009). The Laponite cross-linker has a negative charge, which allows them to adsorb cationic molecules out of solution (Thomas, et. al. 2011; Gregory, et. al. 2013). Past experiments have utilized this property as a mechanism for separations (Thomas, et. al. 2011) or for remote-controlled volume phase transition induction (Gregory, et. al. 2013). Additionally, non-PNIPAM hydrogels have been shown capable of binding to primary antibodies (Massad-Ivanir, et. al. 2010). Our experiments combine these two capabilities by allowing the hydrogels to bind specific cationic molecules, which contain the functional groups necessary for primary antibody conjugation.

Experimental Methodologies

PNIPAM Hydrogel Synthesis

Glass tubes of approximately 1 cm in length with an inner diameter of 1.2 mm were made hydrophobic by submerging them in solution of distilled water, 1.0% volume acetic acid (Sigma Aldrich), and 0.5% volume dimethoxysilane (Sigma Aldrich). After the tubes were allowed to soak in the solution for approximately 15 minutes, the tubes were removed and dried under vacuum in a vacuum oven at 250 oC until dry (approximately 30 minutes).

The protocol for synthesizing PNIPAM hydrogels is also described elsewhere (Arora, et. al. 2009). 40 mg of Laponite-XLG (Southern Clay) was added to 28.5 mL of distilled water and stirred for approximately 30 minutes until the solution returns to clear. 3.5 g of PNIPAM (Sigma Aldrich) is added to the solution and is stirred for approximately 15 minutes until the solution returns to clear. A solution of 30 mg of potassium persulfate dissolved in 1.5 mL of distilled water was added to the stirring PNIPAM and water solution, followed immediately by 24 μL of N,N,N’,N’-tetramethylethylenediamine. The hydrophobic glass tubes were added to the resulting solution, which was then allowed to polymerize for 24 hours at 50 oC.

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After polymerization, the cylindrical hydrogels were removed from the glass tubes and allowed to hydrate in distilled water for 24 hours at room temperature.

Acriflavinium Chloride Adsorption

PNIPAM hydrogels were submerged in a 5 mg mL-1

solution of acriflavine (Sigma Aldrich, 33% acriflavinium chloride) and distilled water for 5 minutes (Figure 1). Longer exposure to acriflavine dye would result in degradation of the hydrogel. After dye adsorption, the hydrogels were rinsed with and submerged in fresh distilled water solutions repeatedly until the un-adsorbed dye had been removed from the hydrogel.

Primary Antibody Attachment

The primary antibody attachment protocol was designed based on protein crosslinking techniques from Hermanson (2008). Primary antibodies for E. coli (KPL) were suspended in PBS 1x to a 20 μg mL -1

concentration. One mL aliquots and 10 μL of glutaraldehyde were added to a separate vial. After 10 seconds, a hydrogel of approximately 1 cm of length and 4 mm in diameter that had adsorbed acriflavinium chloride was added to the vial and allowed to react on a nutator with the glutaraldehyde and primary antibodies for 2 hours at 4 oC. After the primary antibody conjugation was complete, the hydrogels were place in a 1:9 horse serum to PBS 1x solution and allowed to rinse on a nutator for 30 minutes. Three subsequent equivalent rinses were performed, after which the hydrogel was placed in fresh PBS 1x and kept refrigerated.

This protocol was repeated as stated for the attachment of Cryptosporidium (KPL) primary antibodies.

Primary Antibody Attachment Verification

The secondary antibody (KPL) to the primary E. coli antibodies, which were labeled with Alexa 647 fluorescent dye molecules, were added to PBS 1x in a 1:500 secondary antibody to PBS concentration. The primary antibody-functionalized hydrogels were exposed to a 1 mL aliquot of the secondary antibody solution on a nutator for 3 hours at room temperature, with minimal exposure to light. After the secondary antibody exposure, the hydrogels were placed in clean PBS 1x and allowed to rinse on the nutator for 30

Figure 1 Representation of cylindrical hydrogel submersion in an acriflavine dye and water solution. Acriflavine dye contains charged acriflavinium chloride molecules (shown) which are adsorbed by the hydrogel

Hydrogel

Figure 2 Representation showing a primary antibody being conjugated to a hydrogel that has adsorbed acriflavinium chloride by amine-to-amine chemistry. The black line indicates the unknown mechanism (Hermanson 2008) of amine chemistry performed by the homobiofunctional crosslinker glutaraldehyde

Hydrogel

NH2NH2

NH2

NH2

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minutes. After rinsing, the hydrogels were placed in fresh PBS 1x. Three subsequent rinses were performed, after which the hydrogel was placed in fresh PBS 1x and kept refrigerated and away from light.

The protocol was repeated as stated for the verification of Cryptosporidium primary antibodies. Secondary antibodies (Abcam) that correspond to the Cryptosporidium primary antibodies were used in place of the secondary antibodies for E. coli antibody verification.

Four samples were imaged using a 3024 Nikon A1R Multi-Photon Upright Confocal microscope. Three of the samples were cross-sections of hydrogels that went through the acriflavinium chloride adsorption, E. coli or Cryptosporidium primary antibody attachment protocols and were exposed to the corresponding fluorescent-labeled secondary antibodies. The other three samples were the respective controls for the three antibody-conjugated hydrogels. The controls were a necessity to ensure that the presence of secondary antibodies was indicative of primary antibody attachment and not due to secondary antibodies sticking to the structure of the hydrogel or acriflavinium hydrochloride. A summary of the imaged samples can be found in Table 1.

Table 1: Summary of four samples imaged for primary antibody verificationSample 1 Sample 2 Sample 3 Sample 4

AcriflaviniumChloride

Yes Yes Yes Yes

Primary Antibody None Goat Anti-E. coli

None Mouse Anti-Cryptosporidium

Secondary Antibody

Donkey Anti-Goat

Donkey Anti-Goat

Goat Anti-Mouse

Goat Anti-Mouse

Results

Acriflavine Adsorption

Hydrogels have previously been shown to adsorb water-soluble cationic dyes out of solution. (Thomas et. al. 2011; Gregory et. al. 2013). Acriflavine dye contains acflavinium chloride (Figure 1), which has a cationic quaternary amine in its chemical structure. The hydrogels effectively adsorbed the acriflavine dye out of solutions. The acriflavine dye solution contains approximately 33% of the cationic molecule, resulting in leaching of molecules that are not adsorbed by the hydrogel. However, after washing the hydrogels repeatedly to remove un-adsorbed dye, a sufficient amount of acriflavinium chloride is adsorbed in to the structure of the hydrogel (Figure 4).

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 3 Representation of the mechanism for primary antibody attachment, which is the specific binding of fluorescent-labeled secondary antibodies

Hydrogel

Alexa 647 label

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

Figure 4 PNIPAM hydrogels; one has not adsorbed acriflavine dye (top), one after acriflavine dye adsorption (bottom)

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Primary Antibody Attachment

Table 2: Results from Primary Antibody Attachment and Verification Experiment

Sample 1

E. Coli Antibody Control

The picture on the left shows fluorescence as excited by a 488 nm laser. The green fluorescence is indicative of acriflavinium chloride adsorption. The picture on the right shows the lack of fluorescence excited by a 640 nm laser (brightness enhanced by 50%). The lack of fluorescence at 640 nm shows that the Alexa-647-labeled secondary antibodies are not binding to the sample due to the structure of the hydrogel or the presence of acriflavinium chloride.

Sample 2

E. Coli Antibody Sample

The picture on the left shows fluorescence as excited by a 488 nm laser. The green fluorescence is indicative of acriflavinium chloride adsorption. The picture on the right shows the presence of Alexa-647-labled secondary antibodies as excited by a 640 nm laser (brightness enhanced by 50%). The presence of secondary antibodies in Sample 2 but not in Sample 1 shows that the secondary antibodies are present due to the successful conjugation of primary antibodies.

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Sample 3

Cryptosporidium Control

The picture on the left shows fluorescence as excited by a 488 nm laser, which is indicative of acriflavinium chloride adsorption. The picture on the right shows the lack of fluorescence excited by a 640 nm laser (brightness enhanced by 50%). The lack of fluorescence at 640 nm shows that the Alexa-647-labeled secondary antibodies are not binding to the sample due to the structure of the hydrogel or the presence of acriflavinium chloride.

Sample 4

Cryptosporidium Sample

The picture on the left shows fluorescence as excited by a 488 nm laser. The green fluorescence is indicative of acriflavinium chloride adsorption. The picture on the right shows the presence of Alexa-647-labled secondary antibodies as excited by a 640 nm laser (brightness enhanced by 50%). The presence of secondary antibodies in Sample 2 but not in Sample 1 shows that the secondary antibodies are present due to the successful conjugation of primary antibodies.

Discussion

The results of the experiment were successful and have two-fold ramifications. First, the ability to functionalize the PNIPAM hydrogels with the ability to capture microbials is a step towards creating a mobile hydrogel-based biosensor. Hydrogels have previously been shown to capture E. Coli (Massad-Ivanir, et. al. 2010), but this is the first time that PNIPAM, Laponite cross-linked hydrogels have been functionalized to do so. This is important because these hydrogels have durability (Yeghiazarian 2005) and are capable of mobility in both constrained and unconstrained environments via peristaltic locomotion (Gregory 2013), which increases this particular hydrogel’s propensity for usage as a mobile biosensor capable of gathering detection data useful for dynamic mapping of microbe transport. Furthermore, our method of primary antibody conjugation was direct – conjugating antibodies with the homobiofunctional crosslinker glutaraldehyde to dye molecules adsorbed into the hydrogel. This is an alternate method to those performed by Massad-Ivanir et. al. (2010).

Additionally, this experiment has ramifications across the increasingly popular field of hydrogels. In functionalizing the hydrogel by conjugating antibodies to an adsorbed molecule, we have created a new

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technological platform in which any number of molecules or devices may be attached to a hydrogel by first adsorbing a cationic dye with functional groups conducive to attaching the respective molecule or device. Outside the field of biosensors, there are opportunities to increase the usage of hydrogels as a soft biomedical soft tool used for targeted drug delivery or cavity exploration.

Acknowledgements

We would like to thank the National Science Foundation’s Research Experience for Undergraduates program (NSF Type-1 STEP Grant, Grant ID No.: DUE-0756921) as well as the National Science Foundation’s “EAGER: Monitoring Our Nation’s Waters – Towards a Swimming Biosensor to Dynamically Map Microbial Contamination” grant (NSF CBET-1248385) for financial support of this project.

Additionally, we would like to thank the Cincinnati Children’s Hospital and Medical Center Imaging Center, the director of the imaging center Dr. Matthew Kofron, and the manager of the imaging center Mr. Michael Muntifering for the access to and their assistance with fluorescent imaging on the 3024 Nikon A1R Multi-Photon Upright Confocal microscope.

References

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2. <http://www.cdc.gov/nceh/vsp/training/videos/transcripts/water.pdf>

3. <http://www.antimicrobialresistance.dk/data/images/protocols/e%20coli%20methods.pdf>

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5. Thomas, P. C., Cipriano, B. H., and Raghavan, S. R. (2011). “Nanoparticle-crosslinked hydrogels as a class of efficient materials for separation and ion exchange.” Soft Matter, 7(18), 8192–8197.

6. Gregory, J., Riasi, M. S., Cannell, J., Arora, H., Yeghiazarian, L., Nistor, V. (2013). “Remote-Controlled Peristaltic Locomotion in Free-Floating PNIPAM Hydrogels.” Advanced Materials, Submitted for Publication.

7. Massad-Ivanir, N., Shtenberg, G., Zeidman, T., and Segal, E. (2010). “Construction and Characterization of Porous SiO2/Hydrogel Hybrids as Optical Biosensors for Rapid Detection of Bacteria.” Advanced Functional Materials, 20(14), 2269–2277.

8. Hermanson, G. T. (2008). “Homobiofunctional Crosslinkers.” Bioconjugate Techniques, 2nd Edition. Thermofisher Scientific, Rockford, IL, 234-275.

9. Yeghiazarian, L., Mahajan, S., Montemagno, C., Cohen, C., and Wiesner, U. (2005). “Directed Motion and Cargo Transport Through Propagation of Polymer-Gel Volume Phase Transitions.” Advanced Materials, 17(15), 1869–1873.

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